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Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein–protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon proteins. Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organization in plants remain largely unknown. Recently, novel ER network-shaping proteins called Lunapark (LNP) have been identified in yeast and mammalian cells. Here we identify two Arabidopsis LNP homologues and investigate their subcellular localization via confocal microscopy and potential function in shaping the ER network using protein–protein interaction assays and mutant analysis. We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels cisternae in the ER network, whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and lnp1 and lnp1lnp2 amiRNA lines display a reduction in cisternae and larger polygonal areas. Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the network morphology of the plant ER, possibly by regulating the formation of ER cisternae.

In plant cells, molecular connections link the cell wall–plasma membrane–actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)–fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP–Lifeact-labelled network that appeared much more sparse with thick bundles that underwent ‘simple movement’, in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, ‘pairwise image subtraction’, was applied to measurement of the more rapidly remodelling actin network labelled with GFP–FABD2, while the second, ‘cumulative fluorescence intensity’, was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP–Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall–plasma membrane–actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon family of proteins. Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organisation in plants still remain largely unknown. Recently novel ER network-shaping proteins called Lunapark have been identified in yeast and mammalian cells. Here we identify two Arabidopsis LNP homologues and investigate their subcellular localisation via confocal microscopy and potential function in shaping the ER network using protein-protein interaction assays and mutant analysis. We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels the three-way junctions (trivia) of the ER network whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and an lnp1lnp2 amiRNA line displays a less structured ER network. Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the dynamic morphology of the plant ER, possibly by regulating the formation of ER cisternae.

The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture, and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. Using bioinformatic, biochemical, and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with ZmSUN2 in vivo. FRAP experiments using domain-deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research. Footnotes * revised resubmission, oct 3 2018, revised or new text highlighted

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. Recent models suggest that for these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited Airyscan confocal microscopy. In addition, single particle tracking reveals that these proteins move at different rates within the membrane. We show that the actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by affecting single particle dynamics of two proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for differentially regulating protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.